Synaptic changes in the hypothalamic arcuate nucleus of old male rats

The hypothalamic arcuate nucleus (ARCN) was examined ultrastructurally in young (90 to 100 days of age) and old (720 to 930 days of age) male rats. Axodendritic (ADSs) and axosomatic synapses (ASSs) were counted in a field of 18,000 μm² of the ARCN in each brain. In old male rats, the mean numbers of ADSs and ASSs were reduced to approximately 51 and 57% of those of young rats, respectively. Neuronal cell bodies in the ARCN of old male rats were significantly decreased in number compared with those in young male rats. In the ARCN of old animals, increasing numbers of large spherical vesicles or large granular vesicles coexistent with small spherical vesicles were detected in the axon terminals. In some cases, dense inclusion bodies or membranous multilamellar bodies were encountered in the nerve endings. Synaptic thickenings of old male rats were thin, short, and/or discontinuous. As for the dendrites in old male rats, irregular, electron-dense dendritic shafts with a paucity of neurotubules were often observed. Because the ARCN is considered to be one of the principal neural components which regulate secretory functions of the anterior pituitary, loss in the number of synapses and some pathologic changes in its neuropil during the process of aging may apparently impair its integrative control mechanisms. These age-related changes in the ARCN seem to correlate with the aging of the neuroendocrine system.     

Diffusional barrier around the hypothalamic arcuate nucleus in the rat

The contour lines of horseradish peroxidase injection sites in the ventrobasal hypothalamus were distorted by the border between arcuate and ventromedial nuclei as well as between arcuate nucleus and median eminence. The dense array of tanycyte processes is assumed to isolate the arcuate nucleus from the neighboring territories by establishing a diffusional barrier surface.     

ACTH-immunoreactive buotons form synaptic contacts in the hypothalamic arcuate nucleus of rat: evidence for local opiocortin connections

ACTH-like immunoreactive structures were localized in rat hypothalamic arcuate nucleus using the unlabeled antibody, peroxidase-antiperoxidase method. At the ultrastructural level, immunoreactive presynaptic nerve terminals were observed forming symmetrical synaptic contacts with unlabeled dendrites and with ACTH-like immunoreactive perikarya. The results obtained are consistent with the hypothesis that ACTH in the brain acts as a synaptic regulator or transmitter, contributing not only to long projection pathways but also to a local circuit in the arcuate nucleus where ACTH cell bodies are localized.     

The hypothalamus exhibits electrophysiologic evidence for morphine tolerance and dependence

The electrophysiologic effects of morphine (10 mg/kg) were studied in naive rats and after chronic morphine treatment for 5 days. Spontaneous multiunit activity was recorded from the ventromedial hypothalamus of unanesthetized, freely behaving rats implanted previously with permanent electrodes. In the naive condition, firing rates were altered by morphine in 60 of 73 multiunits. After development of tolerance, 28 of multiunits responded to the morphine challenge dose, in 22 of which the direction of change was opposite that observed in the naive condition. The other six were not affected by morphine on day 1 of the experiment and became sensitive to the morphine challenge dose only after the animal had become physically dependent on morphine. Predrug baseline activity in morphine physically dependent rats was altered in 23 recordings compared to the predrug activity obtained in morphine naive animals. Based on effects of morphine and naloxone, responses could be grouped into six electrophysiologic patterns which provide for separation of tolerance from the dependence phenomena.     

Melanocortin-3 receptor mRNA expression in pro-opiomelanocortin neurones of the rat arcuate nucleus

The melanocortins alpha- and gamma-melanocyte-stimulating hormones (alpha- and gamma-MSH) derive from the pro-opiomelanocortin (POMC) precursor. Melanocortins exert a wide range of biological activities in the brain through activation of at least three distinct melanocortin receptor (MC-R) subtypes. In order to determine whether POMC neurones can modulate their own activity, we looked for the possible expression of the MC3-R gene in POMC-positive cell bodies in the rat hypothalamus. In situ hybridization experiments revealed that the density of MC3-R mRNA is particularly high in the arcuate nucleus which contains the main population of POMC neurones in the brain. The occurrence of MC3-R mRNA in POMC-positive cell bodies was demonstrated using a double-labelling in situ hybridization technique. The proportion of POMC neurones expressing MC3-R mRNA was significantly higher in the most rostral (43.5%) than in the most posterior part of the arcuate nucleus (8.2%). These results indicate that melanocortins likely exert a direct regulatory feedback on POMC neurones through activation of MC3-R receptors. Our data also suggest that MC3-R may be involved in the neuroendocrine responses induced by centrally administered melanocortins.     

Estrogen receptors in the medial basal hypothalamus of the rat following complete hypothalamic deafferentation

This study was designed to investigate the effect of complete hypothalamic deafferentation (CHD) on the estrogen receptor (ER) concentration in the MBH as well as LH and FSH secretion. Adult female rats underwent CHD using a Halasz-Pupp knife. Sham CHD and intact animals served as controls. Five days after CHD all the rats were ovariectomized and 2 days later they were decapitated and trunk blood collected and the plasma analysed for LH and FSH by radioimmunoassay (RIA). The brains were rapidly removed and the MBH and preoptic area (POA) were dissected. Brain tissues were homogenized in 2.0 ml of phosphate buffer, centrifuged and the supernatant (cytosol) withdrawn. The cytosols were then incubated at 0–4°C for 3 h with [³H]E₂ or [³H]E₂ + unlabeled E₂. Bound and free receptor was separated using 5–30% sucrose gradient centrifugation, Sephadex LH-20 column or hydroxylapatite receptor assays. CHD resulted in a significant (P < 0.05) reduction in the concentration of ER in the MBH when compared with controls. ER concentration in the POA of the CHD group was not significantly different from the control group. CHD also resulted in a significant (P < 0.005) reduction in the plasma concentration of LH and FSH when compared with the controls. These data suggest that the estrogen receptors in the MBH are influenced by the connections with extrahypothalamic regions and that the effect of hypothalamic deafferentation on gonadotropin secretion may be in part secondary to this reduction of receptors in the MBH.     

Synapse formation of the hypothalamic arcuate nucleus during post-natal development in the female rat and its modification by neonatal estrogen treatment

(1) Semi-quantitative studies on synaptic structures in the arcuate nucleus (ARCN) of female rats were performed by counting synapses in 18,000 μm² area in the middle part of the ARCN in each brain. (2) In 5-day old rats, the neuropil was in an extremely immature state, which was characterized by the presence of extensive extracellular space. Axodendritic and axosomatic synapses at this age were very few in number. In 20-day old rats, the extracellular space was fully occupied by axons, their terminals, dendrites and glial processes. The numbers of axodendritic and axosomatic synapses were increased to more than four and five times those of 5-day old rats, respectively. (3) At 31 days of age, a further increase in the number of synapses was observed. Although the neuropil still contained a mixed population of morphologically immature and mature synapses at this age, the ultrastructural features of the ARCN approached closely those of the adult. (4) The mean numbers of axodendritic and axosomatic synapses at 150 days of age were more than 16 and 17 times those of 5-day old rats. (5) In 6-day old rats which had been treated with estradiol benzoate (EB) from days 1 to 5, the extracellular space in the neuropil matrix was considerably reduced, compared with that of the control. (6) This tendency was much pronounced in 11-day old rats injected with EB from days 1 to 10. (7) In 31-day old rats receiving EB injections for the first 30 days of life, the mean number of axodendritic synapses in the same field area of the ARCN was 2.7-fold greater than that of the controls. As for the axosomatic synapses, there was no significant difference between the estrogenized and controls. The morphologically mature synapses were much more frequently encountered in the estrogenized rats. (8) At 150 days of age, axodendritic synapses did not show a further increase in number and the mean number was not significantly different from those of 31-day old estrogenized and 150-day old control rats. The mean number of axosomatic synapses was also not significantly different from that of the controls. (9) These results suggest that estrogen may have a facilitatory influence on the development of synaptic structures in the ARCN when it is given early postnatally.     

Multiple hypothalamic sites control the frequency of hippocampal theta rhythm

Stimulation of a neural pathway originating in the brainstem reticular formation, with synapses in the medial hypothalamus, activates the hippocampal theta rhythm. The frequency of reticular-elicited theta is determined in the medial supramammillary nucleus (mSuM) completely in anaesthetised rats, but only partially when the animal is awake. We tested other medial hypothalamic sites for their capacity to control theta frequency in awake rats. Blockade of sodium channels (1 microl fast infusion of the local anaesthetic procaine, experiment 1) or increased inhibition by GABA (Chlordiazepoxide [CDP], experiment 2) was found to reduce or increase the frequency of reticular-elicited theta, depending on the precise site of injection, in the region of the dorsomedial hypothalamic nucleus (DMH) and the posterior hypothalamic nucleus (PH). A band of null sites for CDP was located in the region of the ventral border of PH and dorsal border of mSuM. Using 0.5 and 1 microl CDP, and slow infusions (experiment 3), it was found that effective PH sites were also separate from mSuM in the rostrocaudal direction. In experiment 4, the DMH/PH region was mapped with unilateral and bilateral slow infusions of 0.5 microl CDP. CDP significantly reduced frequency in medial (periventricular) and dorsal PH, but not DMH. Bilateral injections appeared to generally sum the usual effects of unilateral injection, producing effects of intermediate size. However, the absolute frequency change in any given site, or with any pair of sites, did not exceed 1 Hz, which is similar to what is seen with single injections in mSuM. Overall, it appears that at, any one time, theta frequency may be determined by a complex interplay between distinct but interacting modulatory regions in the medial hypothalamus.     

Adrenaline concentration and turnover in the arcuate nucleus and median eminence during the critical period in the rat

Previous work has shown a relatively high turnover of adrenaline in the mediobasal hypothalamus during the critical period (15.00-17.00 h) of the proestrous rat. We now report that this high level of adrenergic activity can be detected in the median eminence (turnover rate 1.62 +/- 0.36 pg/micrograms protein/h) rather than the arcuate nucleus (turnover rate 0.18 +/- 0.32 pg/micrograms protein/h). In addition the median eminence was isolated as medial and lateral components and determination of catecholamine concentrations revealed a greater proportion of adrenaline (A) (59%) in the lateral median eminence whereas a larger proportion of dopamine (60%) was found in medial median eminence.     

Expression of mRNA for prolactin receptor (long form) in dopamine and pro-opiomelanocortin neurones in the arcuate nucleus of non-pregnant and lactating rats

Under most conditions, prolactin secretion from the pituitary gland is subject to negative-feedback regulation. Prolactin stimulates dopamine release from tuberoinfundibular (TIDA) neurones in the arcuate nucleus of the hypothalamus, which in turn suppresses the production of prolactin. However, during late pregnancy and continuing into lactation, this feedback mechanism becomes less responsive to prolactin and, as a result, a hyperprolactinaemic state develops. We investigated whether long-form prolactin receptor (PRL-R(L)) mRNA is present on TIDA neurones in nonpregnant and lactating rats. In addition, we examined whether PRL-R(L) mRNA is colocalized on hypothalamic pro-opiomelanocortin (POMC) neurones. Dual-label in situ hybridizations using an (35)S-labelled cRNA probe specific for long-form PRL-R, together with a digoxigenin-labelled RNA probe that encoded either tyrosine hydroxylase (TH) or POMC mRNA, were performed on brain sections. In both nonpregnant and lactating rats, the majority of TH mRNA-positive cells (> 90%) were found to express long-form PRL-R mRNA. In sections from nonpregnant rats, few non-TH positive cells expressed PRL-R(L) mRNA. By contrast, during lactation, the proportion of PRL-R(L) mRNA-positive cells that were not TH mRNA-positive increased to approximately 70%. Only a small number of neurones in this subpopulation of PRL-R(L) mRNA-positive neurones were found to be positive for POMC mRNA. These data show that the loss of responsiveness to prolactin occurring during lactation is not due to down regulation of long-form PRL-R gene expression on TIDA neurones. Moreover, the persistent expression of PRL-R(L) in arcuate neuroendocrine circuits suggests that PRL-R-mediated signalling continues to be important in these neurones during lactation.     

Morphologic evidence that neurokinin B modulates gonadotropin-releasing hormone secretion via neurokinin 3 receptors in the rat median eminence

Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual-label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin-releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB-immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH-ir somata exhibited NK3R staining. ProNKB and NK3R-ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB-ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis.     

Recovery of sexual receptivity in female rats with lesions of the ventromedial hypothalamus

Bilateral lesions of the hypothalamic ventromedial nucleus (VMN) effectively eliminated the induction by estrogen-progesterone of lordosis behaviors 4 to 6 weeks after the brain surgery. However, recovery of the lordosis response was clearly evident in the rats with effective VMN lesions 2 weeks after the first test. This suggests that the VMN may not be essential to the expression of hormonally induced lordosis behavior, but may be involved in a supplemental lordosis mediating mechanism.     

The projections of the dorsomedial hypothalamic nucleus in the rat

The dorsomedial hypothalamic nucleus (DMH) output pathways are revealed by using autoradiographic tracing of tritium labeled Leucine and by the recently introduced Phaseolus vulgaris leuco-agglutinin immunocytochemical method. Terminal labeling appears in the dorsal motor nucleus of the vagus, nucleus ambiguus and in the parvocellular reticular formation at the lower medullary level. Mesencephalic labeling is found in the periaqueductal gray at the level of the oculomotor nucleus. In the hypothalamus labeled terminal boutons are identified in the lateral and ventromedial hypothalamic nuclei but also in the parvocellular paraventricular nucleus. Furthermore, the circumventricular organs are found to receive a dense DMH input, particularly the organum vasculosum of the lamina terminalis and the subfornical organ. These findings are discussed in relation to the dorsomedial nucleus involvement in the control of feeding and pancreatic hormone release. It appears that the DMH participates in this control via descending pathways to the preganglionic pancreas innervating neurons but also via a neuroendocrine route. The latter connection is indicated by terminal labeling in the parvocellular paraventricular nucleus in the area that contains the corticotropin-releasing factor positive cells.     

Distribution of prolactin receptor immunoreactivity in the brain of estrogen-treated,ovariectomized rats

Although there is extensive evidence for effects of prolactin (PRL) on the brain, knowledge about the PRL receptor (PRL-R) in the brain is limited. By using monoclonal antibodies raised against purified rat liver PRL-R, the distribution of PRL-R was investigated by immunohistochemistry in brains of the estrogen-treated ovariectomized (OVX+E) rat and the adult male rat. Immunohistochemistry was performed by using the avidin biotinylated horse radish peroxidase macromolecular complex method. In both male and OVX+E rats, strong immunostaining was detected in the choroid plexus of all cerebral ventricles. This immunostaining was localized predominately on epithelial cell membranes. In the OVX+E female rat, scattered immunoreactive perikarya were observed in the arcuate nucleus, periventricular hypothalamic nucleus, preoptic area, suprachiasmatic nucleus, and supraoptic nucleus of the hypothalamus. Immunostaining in hypothalamic nuclei was localized on neuronal cell bodies as well as on neuronal processes. In addition, there was extensive PRL-R immunoreactivity throughout the globus pallidus and ventral pallidum. Immunostaining in these striatal regions was not associated with neuronal cell bodies but appeared to be localized on processes or glial cells. In the male rat, less immunostaining was observed in the hypothalamus, and there was no immunostaining in the corpus striatum. No significant staining was observed in the cerebral cortex, thalamus, or hindbrain of either male or OVX+E rats. The implication of PRL-R existence in these brain regions remains to be investigated. J. Comp. Neurol. 394:462–474, 1998. © 1998 Wiley-Liss, Inc.     

Galanin immunoreactivity in hypothalamic neurons: further evidence for multiple chemical messengers in the tuberomammillary nucleus

By using a specific antibody against the 29 amino-acid peptide galanin (Gal) with light and electron microscopic immunocytochemistry, we have studied the distribution of Gal immunoreactivity in the posterior hypothalamic magnocellular neurons of the rat. In colchicine-treated rats, a large number of Gal-immunoreactive cells were identified within all subdivisions of the tuberomammillary nucleus. The majority of these cells are large multipolar or fusiform neurons, with long, sparsely branching dendrites. A small number project to the ventral hippocampus, as shown by experiments with the retrograde tracing of Fast Blue. Ultrastructural examination of the Gal-immunoreactive cells confirms their indentity as magnocellular neurons, with dense deposits of immunoreaction product, particularly in small ribosomal arrays and in large, dense-cored vesicles. Axosomatic synapses occur on these neurons. The axonal boutons synapse with asymmetric and symmetric junctions and contain small synaptic vesicles as well as numerous large, dense-cored vesicles, which display Gal immunoreactivity. Sequential staining of thin, alternate sections with antibodies against Gal and L-histidine decarboxylase (HDCase; EC 4.1.1.22) showed colocalization of Galand HDCase-immunoreactivities in a majority of tuberomammilary neurons. The finding of Gal immunoreactivity within histamine-producing neurons of the tuberomammillary nucleus adds to the multiplicity of potential neuronal messengers utilized by these cells.     

Effects of adrenalectomy on CART expression in the rat arcuate nucleus

In order to test for glucocorticoid regulation of CART in the arcuate nucleus, adrenalectomies (ADX) and hormone replacements (HRs) were carried out in groups of rats. CART mRNA levels were determined by in situ hybridization and CART peptide levels by immunocytochemistry. ADX caused a lowering of CART mRNA and peptides levels in the arcuate and this was reversed by HR. These results indicate a glucocorticoid regulation of CART in the arcuate. The regulation could be direct through an action of glucocorticoid receptors or indirectly through ADX-induced changes in leptin levels. These findings suggest a role for CART in the stress response.